Mechanism for Activation of the EGF Receptor Catalytic Domain by the Juxtamembrane Segment

Jura, Natalia; Endres, Nicholas; Engel, Kate; Deindl, Sebastian; Das, Rahul Peter; Lamers, Meindert H.; Wemmer, David E.; Zhang, Xuewu; Kuriyan, John

doi:10.1016/s9999-9994(09)20407-7

Cited by 96 publications

(185 citation statements)

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“…Structural modeling of the intracellular kinase domain region based on a published HER3 kinase model did not reveal major structural differences between wild type HER3, HER3-2-3, and HER3-2-2 (Jura et al, 2009b;Jura et al, 2009a) (Fig. 5).…”

Section: Discussionmentioning

confidence: 96%

“…Dimerization among the EGFR family of receptors leads to allosteric activation of the kinase domains of the partners, and the activated kinase domains in turn phosphorylate the tyrosine residues in the C-terminus of the receptors (Jura et al, 2009b;Jura et al, 2009a). HER3 is different from EGFR, HER2 and HER4 in that it lacks intrinsic kinase activity and has not been reported to form homodimers on cell surface (Sierke et al, 1997;Berger et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

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HER3 intracellular domains play a crucial role in HER3/HER2 dimerization and activation of downstream signaling pathways

et al. 2012

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Dimerization among the EGFR family of tyrosine kinase receptors leads to allosteric activation of the kinase domains of the partners. Unlike other members in the family, the kinase domain of HER3 lacks key amino acid residues for catalytic activity. As a result, HER3 is suggested to serve as an allosteric activator of other EGFR family members which include EGFR, HER2 and HER4. To study the role of intracellular domains in HER3 dimerization and activation of downstream signaling pathways, we constructed HER3/HER2 chimeric receptors by replacing the HER3 kinase domain (HER3-2-3) or both the kinase domain and the C-terminal tail (HER3-2-2) with the HER2 counterparts and expressed the chimeric receptors in Chinese hamster ovary (CHO) cells. While over expression of the intact human HER3 transformed CHO cells with oncogenic properties such as AKT/ERK activation and increased proliferation and migration, CHO cells expressing the HER3-2-3 chimeric receptor showed significantly reduced HER3/HER2 dimerization and decreased phosphorylation of both AKT and ERK1/2 in the presence of neuregulin-1 (NRG-1). In contrast, CHO cells expressing the HER3-2-2 chimeric receptor resulted in a total loss of downstream AKT activation in response to NRG-1, but maintained partial activation of ERK1/2. The results demonstrate that the intracellular domains play a crucial role in HER3's function as an allosteric activator and its role in downstream signaling.

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Section: Discussionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

HER3 intracellular domains play a crucial role in HER3/HER2 dimerization and activation of downstream signaling pathways

et al. 2012

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“…However, these systems are not compatible with affinity purification because they were designed to observe natural interactions between protein subunits, but not to maintain them. Another coiled-coil pair, GCN4, has been used as a tool for the purification of VEGF receptor ectodomains 30 and in EGFR kinase domain studies, 31 but it is based on the natural activator GCN4 and thus may interact with endogenous proteins. We wanted to generate a completely synthetic pair to avoid any potential interactions with cellular proteins.…”

Section: Discussionmentioning

confidence: 99%

Induced heterodimerization and purification of two target proteins by a synthetic coiled‐coil tag

Fernández-Rodríguez

Marlovits

2012

Protein Science

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A synthetic de novo designed heterodimeric coiled-coil was used to copurify two target fluorescent proteins, Venus and enhanced cyan fluorescent protein (ECFP). The coiled-coil consists of two 21-amino acid repetitive sequences, (EIAALEK) 3 and (KIAALKE) 3 , named E3 and K3, respectively. These sequences were fused to the C-termini of ECFP or Venus followed by either a strep-or a his-tag, respectively, for affinity purification. Mixed lysates of Venus-K3 and ECFP-E3 were subjected to consecutive affinity purification and showed highly specific association between the coiled-coil pair by SDS-PAGE, gel filtration, isothermal titration calorimetry (ITC), and fluorescence resonance energy transfer (FRET). The tagged proteins eluted as heterodimers at the concentrations tested. FRET analysis further showed that the coiled-coil pair was stable in buffers commonly used for protein purification, including those containing high salt concentration and detergent. This study shows that the E3/K3 pair is very well suited for the copurification of two target proteins expressed in vivo because of its high specificity: it forms exclusively heterodimers in solution, it does not interact with any cellular proteins and it is stable under different buffer conditions.

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“…This was not expected as EGFRc958 retains all the extracellular dimerization interfaces found in EGFRwt and also responds in a ligand-induced receptor-mediated dimerization mechanism. This impaired dimerization ability is likely due to the absence of recently described dimerization-related sequences which are absent in the EGFRc958-truncated receptor (21). This detection by PLA of the extracellularly deleted EGFRvIII to dimerize with EGFRwt or EGFRc958 has been of debate and not been resolved with standard techniques to date.…”

Section: Mutant Egfrs Display Aberrant Dimerization Characteristicsmentioning

confidence: 99%

In Situ Analysis of Mutant EGFRs Prevalent in Glioblastoma Multiforme Reveals Aberrant Dimerization, Activation, and Differential Response to Anti-EGFR Targeted Therapy

Gajadhar

Bogdanovic

Muñoz

et al. 2012

Molecular Cancer Research

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Aberrations in epidermal growth factor receptor (EGFR/ErbB1) are the most common oncogenic alterations in glioblastoma multiforme (GBM), the most common primary brain tumor. Interactions between wild-type (wt) and mutant EGFRs and their subsequent activation are of biologic and potential therapeutic importance in GBMs. We recently showed that in situ proximity ligation assay (PLA) allows for quantitative evaluation of EGFR dimerization and activation in intact cells. Using this in situ platform, we show the aberrant homo-/heterodimeric properties of EGFRvIII and EGFRc958 mutants, the two most common EGFR mutants in GBMs. In addition, dimer phosphoactivation status could be detected by PLA with superior signal-noise ratio (>17-fold) and sensitivity (>16-fold) than immunofluorescence-based phospho-EGFR measurements. Dimer activation analysis indicated quantitative activation differences of mutant dimers. These aberrant features were not overexpression dependent but appeared independent of cellular expression levels, suggesting inherent properties of the mutant receptors. Moreover, we observed in situ detection of EGFRwt-EGFRvIII heterodimerization in GBM specimens, supporting our cell line observations. Notably, currently used anti-EGFR therapeutics, such as cetuximab, matuzumab, and panitumumab, could effectively block EGFRwt dimerization and activation but did not equally impair EGFRvIII homodimers, EGFRwt-EGFRvIII, or EGFRvIII-EGFRc958 heterodimers. EGFRvIII appears to have intrinsic phosphoactivation independent of dimerization as matuzumab blockade of homodimerization had no effect on receptor phosphorylation levels. These data suggest differences in the dimerization-blocking efficacy of EGFR monoclonal antibodies as mutant EGFR dimer configurations prevalent in GBMs can evade blockade by anti-EGFR treatments. Further studies are warranted to evaluate whether this evasion contributes to poor therapeutic response or resistance. Mol Cancer Res; 10(3); 428-40. Ó2012 AACR.

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Mechanism for Activation of the EGF Receptor Catalytic Domain by the Juxtamembrane Segment

Cited by 96 publications

References 0 publications

HER3 intracellular domains play a crucial role in HER3/HER2 dimerization and activation of downstream signaling pathways

HER3 intracellular domains play a crucial role in HER3/HER2 dimerization and activation of downstream signaling pathways

Induced heterodimerization and purification of two target proteins by a synthetic coiled‐coil tag

In Situ Analysis of Mutant EGFRs Prevalent in Glioblastoma Multiforme Reveals Aberrant Dimerization, Activation, and Differential Response to Anti-EGFR Targeted Therapy

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